Description:

Workflow begin

This tutorial comes from Seurat

Load required packages

library(dplyr)
library(Seurat)
library(leiden)
library(ggplot2)

Load data

Input is a directory that contains fastq files

previous_time <- proc.time()[3]
Estrus.data <-Read10X(data.dir ="/home/nguyen/Estrus_cellranger_count/Estrus_Raw/")
proc.time()[3] - previous_time
elapsed 
   8.09 
  • View(Estrus.data) - run this command to investigate the output
  • Current dataset:
    • 31253 genes
    • 6599 cells

Initialize the Seurat object

  • Initialize the Seurat object with the raw (non-normalized data).
  • Filter out genes that are detected in less than 3 cells
  • Filter out cells that have less than 200 genes expressed
previous_time <- proc.time()[3]
Estrus <- CreateSeuratObject(counts = Estrus.data, project = "Estrus", min.cells = 3, min.features = 200)
proc.time()[3] - previous_time
elapsed 
  1.876 
Estrus
An object of class Seurat 
19420 features across 6481 samples within 1 assay 
Active assay: RNA (19420 features, 0 variable features)

*Output: + 19420 genes + 6481 cells

Calculate the percentage of mitochondrial genes

  • Add “percent.mt” column to object metadata
  • Percentage of mitochondrial genes is stored in “percent.mt”
  • “^mt-” means strings start with mt (lowercase mt for mice, uppercase MT for human)
Estrus[["percent.mt"]] <- PercentageFeatureSet(Estrus, pattern = "^mt-")
# This is the list of genes that start with mt
mito_genes <- grep("^mt-", rownames(Estrus) , ignore.case=T, value=T)
print(mito_genes)
 [1] "mt-Nd1"  "mt-Nd2"  "mt-Co1"  "mt-Co2"  "mt-Atp8" "mt-Atp6" "mt-Co3"  "mt-Nd3"  "mt-Nd4l" "mt-Nd4"  "mt-Nd5"  "mt-Nd6"  "mt-Cytb"
# This is the data in "percent.mt" column
percent_mito <- FetchData(object = Estrus, vars = "percent.mt") #how to fetch data
head(percent_mito)
# Visualize QC metrics as a violin plot
VlnPlot(Estrus, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3)

# FeatureScatter is typically used to visualize feature-feature relationships, but can be used
# for anything calculated by the object, i.e. columns in object metadata, PC scores etc.
plot1 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "percent.mt")
plot2 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
plot1 + plot2

Filter out low-quality cells

  • We filter out cells that have unique feature counts over 6000
  • Because any cell that has more than 6000 genes are likely doublets
  • Note: Seurat tutorial used 2500 instead of 6000
  • We also filter out cells that have more than 5% mitochondrial genes
  • Because those are likely to be nonviable cells
previous_time <- proc.time()[3]
Estrus <- subset(Estrus, subset = nFeature_RNA < 6000 & percent.mt < 5)
proc.time()[3] - previous_time
elapsed 
  0.235 
Estrus
An object of class Seurat 
19420 features across 2174 samples within 1 assay 
Active assay: RNA (19420 features, 0 variable features)
  • After filtering step, the dataset is left with
  • 19420 genes
  • 2174 cells

Normalizing the data

  • Perform log-normalization method
  • Feature counts for each cell are divided by the total counts for that cell and multiplied by the scale.factor
  • scale.factor default value = 10,000
  • This is then natural-log transformed using log1p
previous_time <- proc.time()[3]
Estrus <- NormalizeData(Estrus, normalization.method = "LogNormalize", scale.factor = 10000)
Performing log-normalization
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
proc.time()[3] - previous_time
elapsed 
  0.223 

Identification of highly variable features (feature selection)

previous_time <- proc.time()[3]
Estrus <- FindVariableFeatures(Estrus, selection.method = "disp", nfeatures = 5000)
Calculating gene means
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
Calculating gene variance to mean ratios
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
proc.time()[3] - previous_time
elapsed 
    0.4 

Plotting highly variable features

# Identify the 10 most highly variable genes
top10 <- head(VariableFeatures(Estrus), 10)
top10
 [1] "Hbb-bs" "Hba-a1" "Hba-a2" "Dcpp3"  "Hbb-bt" "Ccl21a" "Apoe"   "Cd74"   "Ccl5"   "Dcpp1" 
#{r, fig.width=10,fig.height=11}
#df %>% ggplot(aes(x = x, y = y)) + geom_point()
# Plot variable features with and without labels
plot1 <- VariableFeaturePlot(Estrus)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
When using repel, set xnudge and ynudge to 0 for optimal results
plot1 + plot2 
Warning: Removed 2 rows containing missing values (geom_point).
Warning: Removed 2 rows containing missing values (geom_point).

Regress out data and perform scaling

  • Regressing out total counts per cell and percent.mt before scaling.
  • Take out mitochondrial genes before scaling (analyzing) the data
  • Because mito_genes shouldn’t be accounted for cell population analysis.
previous_time <- proc.time()[3]
all.genes <- rownames(Estrus)
Estrus <- ScaleData(Estrus, features = all.genes, vars.to.regress = c("nCount_RNA","percent.mt"), scale.max = 10)
Regressing out nCount_RNA, percent.mt

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Centering and scaling data matrix

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# Explanations below are from [Seurat](https://satijalab.org/seurat/archive/v3.0/cell_cycle_vignette.html)
# vars.to.regress = RegressOut in Seurat version 1.4 or lower
# However, as the results of this procedure are stored in the scaled data slot (therefore overwriting the output of ScaleData) 
# This functionality is merged into the ScaleData function itself.
proc.time()[3] - previous_time
elapsed 
549.548 

Perform linear dimensional reduction (PCA)

previous_time <- proc.time()[3]
Estrus <- RunPCA(Estrus, features = VariableFeatures(object = Estrus))
PC_ 1 
Positive:  Gm867, 1700016K19Rik, 1700007K13Rik, Ccdc153, Tmem212, Gm19935, Dynlrb2, 1110017D15Rik, Cfap126, Fam166c 
       Fam183b, Vpreb3, Rsph1, Dnali1, Foxj1, Lrrc10b, Sntn, Tctex1d4, 1700024G13Rik, Mlf1 
       Pifo, Dnah12, Prr29, Hdc, 2410004P03Rik, Timp4, Efcab10, Tppp3, 2610028H24Rik, Lrrc51 
Negative:  Rps28, Rps19, Rpl18a, Rpl23a, Rplp0, Rpl13, Rpl37, Rpl32, Rps12, Rps18 
       Rps5, Rps29, Rps24, Rpl37a, Rps7, Rps27a, Rps23, Cd63, Rpl35, Rpl39 
       Rps4x, Rps11, Rpl27a, Rps20, Rps15a, Rps16, Rps3a1, Rpl19, Rpl10, Rps2 
PC_ 2 
Positive:  Cd68, Lcn2, Sprr2f, Arg1, Wfdc2, Sftpd, Pdzk1ip1, Prap1, Cxcl17, Clca1 
       Serpina1e, Fxyd3, Pigr, Ltf, Tmem45b, Ctss, Padi2, Tacstd2, Ly6a, Muc4 
       Padi1, Ckmt1, Trpv6, Fxyd4, Muc1, Pglyrp1, Rnf128, Cndp2, Car2, Alox5ap 
Negative:  Serpinh1, Sparc, Col1a2, Col3a1, Col1a1, Col6a1, Cald1, Bgn, Mgp, Aebp1 
       Col6a2, Igfbp7, Rarres2, Zbtb20, Dcn, Nbl1, Rcn3, Igfbp6, Mdk, Sparcl1 
       Fxyd1, Mfap2, Csrp2, Pbx1, Fxyd6, Col6a3, Col4a1, Gsn, Lgals1, Htra3 
PC_ 3 
Positive:  Tyrobp, Fcer1g, Ctss, Cd52, Csf1r, Cd74, H2-Aa, H2-Ab1, Laptm5, H2-Eb1 
       Aif1, Coro1a, Apoe, C1qc, Lyz2, C1qa, C1qb, H2-DMa, Pld4, Ms4a6c 
       Cx3cr1, Cybb, Ly86, Spi1, Ms4a7, Mpeg1, Trf, Fcgr3, Lst1, Fyb 
Negative:  Plet1, Ovgp1, Krt18, Wfdc18, Spint2, Aldoc, Gstm2, Epcam, Emb, Kctd14 
       Tpd52l1, Clu, Fxyd3, Dcxr, Kcnk1, Hddc2, Agr2, Rbp1, Psmd3, Cd9 
       Cldn22, Cystm1, Cldn7, Ppp1r1b, Gstm1, Rpl41, Mgst1, Tmem218, Krt19, Krt8 
PC_ 4 
Positive:  Clca1, Cxcl17, Pigr, Plac8, Muc4, Prap1, Slc40a1, Ly6a, Serpina1e, Tmem45b 
       Sprr2f, Trpv6, Tacstd2, Tmprss4, Tspan1, Prxl2a, Arg1, Padi2, Fxyd4, Fcgbp 
       Fut9, Lrg1, Ckmt1, Morrbid, Car2, Fbln1, Upk3bl, Ncoa7, Pdzk1ip1, Sult1d1 
Negative:  Kctd14, Gstm2, Ovgp1, Emb, Sdf2l1, Clu, Ctsc, Cbs, Tmem218, Gstm1 
       Slc1a3, Ppp1r1b, Smim14, Lrpap1, Shisa8, Cldn22, Fn1, Agr2, Wfdc18, Rcn1 
       Dkk3, Ncf2, Bex4, Gatm, Ccnd1, Gdpd2, Aldoc, Manf, Selenom, Cyth4 
PC_ 5 
Positive:  Gsn, Pdgfra, Htra3, Mfap4, Dpt, Mfap2, Serpinf1, Ntrk2, Col6a3, Mdk 
       Lpl, Penk, Lama2, Matn2, Col15a1, Gpm6b, Lrp1, Col1a1, Col5a2, Sparcl1 
       Ramp3, Htra1, Ftl1, Spon1, Ramp1, Islr, Col1a2, Plxdc2, Rxfp1, Osr2 
Negative:  Egfl7, Kdr, Ecscr, Cavin2, Pecam1, Cldn5, Cav1, Cdh5, Tie1, Mmrn1 
       Emcn, Flt4, Ushbp1, Mmrn2, Ccl21a, Podxl, Gimap6, S1pr1, Reln, Prox1 
       Adgrf5, Myct1, Gpm6a, Esam, Pard6g, Lyve1, Gimap1, Stmn2, Gimap4, Arhgap29 
proc.time()[3] - previous_time
elapsed 
   5.29 

Examine and visualize PCA results a few different ways

print(Estrus[["pca"]], dims = 1:5, nfeatures = 5)
PC_ 1 
Positive:  Gm867, 1700016K19Rik, 1700007K13Rik, Ccdc153, Tmem212 
Negative:  Rps28, Rps19, Rpl18a, Rpl23a, Rplp0 
PC_ 2 
Positive:  Cd68, Lcn2, Sprr2f, Arg1, Wfdc2 
Negative:  Serpinh1, Sparc, Col1a2, Col3a1, Col1a1 
PC_ 3 
Positive:  Tyrobp, Fcer1g, Ctss, Cd52, Csf1r 
Negative:  Plet1, Ovgp1, Krt18, Wfdc18, Spint2 
PC_ 4 
Positive:  Clca1, Cxcl17, Pigr, Plac8, Muc4 
Negative:  Kctd14, Gstm2, Ovgp1, Emb, Sdf2l1 
PC_ 5 
Positive:  Gsn, Pdgfra, Htra3, Mfap4, Dpt 
Negative:  Egfl7, Kdr, Ecscr, Cavin2, Pecam1 
VizDimLoadings(Estrus, dims = 1:2, reduction = "pca")

DimPlot(Estrus, reduction = "pca") + ggtitle(label = 'Including immune cells')

FeaturePlot(Estrus, reduction = "pca", features = c('Ovgp1', 
                                                    'Foxj1', 'Igf1', 'Pdxk', 
                                                    'Fxyd4', 'Bsg', 
                                                    'Serpina1e', 'Krt19'))

FeaturePlot(Estrus, reduction = "pca", features = c('Dcn', 'Pax8',
                                                    'Ephx2', 
                                                    'Pecam1', 'Pdgfra', 'Myh11', 'Atp2b4', 
                                                    'Cd52'))

Determine the ‘dimensionality’ of the data

  • Seurat tutorial uses JackStraw() and and ScoreJackStraw to determine dimensionality
  • the syntax is:
    • Estrus <- JackStraw(Estrus, num.replicate = 100)
    • Estrus <- ScoreJackStraw(Estrus, dims = 1:20)
  • However, it takes very long.
  • An elbow plot often corresponds well enough with the significant dims and is much faster to run than Jackstraw
  • According to the graph below, choose the first 37 principle components to use
ElbowPlot(Estrus, ndims = 40)

Find neighbors

  • Dims = dimensions = determined as 37 from the above step
previous_time <- proc.time()[3]
Estrus_new <- FindNeighbors(Estrus, dims = 1:37, k.param = 10)
Computing nearest neighbor graph
Computing SNN
proc.time()[3] - previous_time
elapsed 
  0.307 

Find cell clusters using Leiden’s method

# Algorithm = 4 specifies Leiden's method
previous_time <- proc.time()[3]
Estrus_new <- FindClusters(Estrus_new, resolution = 0.12, algorithm = 4)
proc.time()[3] - previous_time
elapsed 
  0.559 

Constructing UMAP plot

previous_time <- proc.time()[3]
Estrus_new <- RunUMAP(Estrus_new, dim = 1:37) 
18:28:21 UMAP embedding parameters a = 0.9922 b = 1.112
18:28:21 Read 2174 rows and found 37 numeric columns
18:28:21 Using Annoy for neighbor search, n_neighbors = 30
18:28:21 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
18:28:21 Writing NN index file to temp file /tmp/RtmpdPzDpp/filec7e289291cc
18:28:21 Searching Annoy index using 1 thread, search_k = 3000
18:28:22 Annoy recall = 100%
18:28:22 Commencing smooth kNN distance calibration using 1 thread
18:28:23 Initializing from normalized Laplacian + noise
18:28:23 Commencing optimization for 500 epochs, with 85588 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
18:28:25 Optimization finished
proc.time()[3] - previous_time
elapsed 
  4.369 

Visualization

DimPlot(Estrus_new, reduction = "umap") + ggtitle(label = 'Including immune cells')

FeaturePlot(Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Dcn', 'Serpina1e', 'Sprr2f',
                                                         'Fxyd4', 'Ly6a', 'Epcam', 'Krt19', 'Vim', 'Pdgfra'))

FeaturePlot(Estrus_new, reduction = "umap", features = c('Myh11', 
                                        'Dkk2', 'Slitrk6', 'Dio2', 'Smoc2', 'Tes', 'Dab1', 'Camk1d', 'Pecam1', 'Cd52','Mki67'))

Removing immune cells

sub_Estrus = subset(x = Estrus_new, ident = c(1,2,3,4,5,7,8,9,10,12))
DimPlot(sub_Estrus, reduction = "umap") + ggtitle(label = 'Immune cells removed')

FeaturePlot(sub_Estrus, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Fxyd4', 'Cxcl17', 'Myh11', 'Cd52', 'Twist2', 'Pecam1'))

FeaturePlot(sub_Estrus, reduction = "umap", features = c('Pdgfra', 'Ephx2', 'Dcn', 'Krt19', 'Epcam', 'Vim','Mki67'))

sub_Estrus
An object of class Seurat 
19420 features across 1925 samples within 1 assay 
Active assay: RNA (19420 features, 5000 variable features)
 2 dimensional reductions calculated: pca, umap
  • After removing immune cells, the dataset is left with:
    • 19420 genes
    • 1925 cells

Re-run analysis after excluding immune cells

previous_time <- proc.time()[3]
sub_Estrus <- RunPCA(sub_Estrus, features = VariableFeatures(object = sub_Estrus))
PC_ 1 
Positive:  Rps28, Rps19, Rplp0, Rpl13, Rpl18a, Rpl23a, Rpl37, Rpl32, Rps18, Rps5 
       Cd63, Rps12, Rps7, Rpl37a, Rps24, Rpl39, Rps27a, Rps23, Rps4x, Rps29 
       Rpl35, Rps20, Rpl27a, Rps3a1, Rps15a, Rps11, Rps2, Rpl10a, Rps16, Rpl10 
Negative:  Gm867, 1700016K19Rik, 1700007K13Rik, Ccdc153, Tmem212, Gm19935, Dynlrb2, Fam166c, 1110017D15Rik, Cfap126 
       Fam183b, Sntn, Foxj1, Dnali1, Vpreb3, Rsph1, Lrrc10b, Tctex1d4, Dnah12, 1700024G13Rik 
       Mlf1, Pifo, Prr29, 2410004P03Rik, Tppp3, Hdc, Dnaja4, Efcab10, Cfap77, Timp4 
PC_ 2 
Positive:  Lcn2, Fxyd3, Ltf, Wfdc2, Sftpd, Arg1, Pdzk1ip1, Sprr2f, Prap1, Epcam 
       Cxcl17, Serpina1e, Clca1, Muc1, Padi1, Rnf128, Pigr, Plet1, Elf3, Tmem45b 
       Ly6a, Tacstd2, Muc4, Ckmt1, Cldn7, Car2, Fxyd4, Pglyrp1, Trpv6, Tspan8 
Negative:  Col1a2, Serpinh1, Aebp1, Col3a1, Cald1, Col6a1, Col1a1, Sparc, Mgp, Rarres2 
       Bgn, Col6a2, Dcn, Rcn3, Igfbp6, Rnase4, Igfbp7, Zbtb20, Lgals1, Mdk 
       Nbl1, Csrp2, Igf1, Fxyd1, Lrp1, Sparcl1, Mfap2, Col6a3, Gsn, Timp2 
PC_ 3 
Positive:  Ovgp1, Kctd14, Gstm2, Emb, Clu, Wfdc18, Gstm1, Tmem218, Cbs, Aldoc 
       Ppp1r1b, Cldn22, Agr2, Slc1a3, Lrpap1, Shisa8, Tpd52l1, Rcn1, Dkk3, Sdf2l1 
       Fn1, Bex4, Manf, Gdpd2, Smim14, Selenom, Kcne3, Enho, Slc15a2, Slco2a1 
Negative:  Plac8, Clca1, Pigr, Muc4, Cxcl17, Slc40a1, Ly6a, Prap1, Tspan1, Tmem45b 
       Trpv6, Tmprss4, Serpina1e, Sprr2f, Tacstd2, Padi2, Prxl2a, Fcgbp, Fbln1, Cfh 
       Fut9, Fxyd4, Arg1, Rasa4, Lrg1, Morrbid, Ncoa7, Ckmt1, Slc39a8, Upk3bl 
PC_ 4 
Positive:  Upk3b, Nkain4, Lrrn4, Upk1b, C2, Myrf, Lgals2, Fgf1, Lgals7, Smoc2 
       Gpm6a, Serpinb6b, Slpi, Cldn15, Ogn, Cybrd1, Plxna4, Rspo1, Nrgn, Msln 
       Aldh1a2, Rpp25, Ptgis, Hpgd, Adam33, Prph, Lmcd1, Tm4sf5, Hacd4, Prss23 
Negative:  Pdgfra, Sparcl1, Gsn, Dpt, Mfap4, Htra3, Mfap2, Col15a1, Itih5, Gpm6b 
       Ramp3, Lama2, Ntrk2, Penk, G0s2, Serpinf1, Lpl, Spon1, Col6a3, Enpp2 
       Rxfp1, Pdgfrl, Matn2, Lhfp, Ctsk, Plpp3, Ramp1, P2ry1, Col5a3, Nrp1 
PC_ 5 
Positive:  Dcn, Aebp1, Ctsl, Pdgfra, Dpt, Pcolce, Fbln1, Lpl, Cygb, Ramp3 
       Col15a1, Ntrk2, Htra1, Meg3, Plxdc2, Htra3, Lum, Col5a3, Efemp1, Rnase4 
       Itm2a, Col3a1, Xdh, Cdh11, P2ry1, Ctsk, C1s1, Itm2b, Col6a2, Dkk2 
Negative:  Myh11, Cnn1, Lmod1, Fbxl22, Acta2, Mustn1, Actg2, Tagln, Sh3bgr, Mcam 
       Des, Mylk, Nexn, Myl9, Ppp1r14a, Slc22a3, Tpm2, Aoc3, Myom1, Synpo2 
       Postn, Carmn, Smtn, Dmpk, Ppp1r12b, Tpm1, Pgm5, Rbpms2, Slc2a4, Pdlim3 
proc.time()[3] - previous_time
elapsed 
  4.379 
DimPlot(sub_Estrus, reduction = "pca") + ggtitle(label = 'Immune cells removed')

FeaturePlot(sub_Estrus, reduction = "pca", features = c('Esr1', 'Ovgp1', 'Foxj1', 'Fxyd4', 'Ephx2', 'Dcn'))

FeaturePlot(sub_Estrus, reduction = "pca", features = c('Fos', 'Kcne3', 'Serpina1e', 'Crabp2', 'Pgr', 'Vim'))

previous_time <- proc.time()[3]
sub_Estrus_new <- FindNeighbors(sub_Estrus, dims = 1:37)
Computing nearest neighbor graph
Computing SNN
proc.time()[3] - previous_time
elapsed 
  0.306 
previous_time <- proc.time()[3]
sub_Estrus_new <- FindClusters(sub_Estrus_new, resolution = 0.045, algorithm = 4)
proc.time()[3] - previous_time
elapsed 
  0.592 
previous_time <- proc.time()[3]
sub_Estrus_new <- RunUMAP(sub_Estrus_new, dim = 1:37)
18:31:18 UMAP embedding parameters a = 0.9922 b = 1.112
18:31:18 Read 1925 rows and found 37 numeric columns
18:31:18 Using Annoy for neighbor search, n_neighbors = 30
18:31:18 Building Annoy index with metric = cosine, n_trees = 50
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
18:31:18 Writing NN index file to temp file /tmp/RtmpdPzDpp/filec7e27d32c007
18:31:18 Searching Annoy index using 1 thread, search_k = 3000
18:31:19 Annoy recall = 100%
18:31:19 Commencing smooth kNN distance calibration using 1 thread
18:31:20 Initializing from normalized Laplacian + noise
18:31:20 Commencing optimization for 500 epochs, with 77854 positive edges
0%   10   20   30   40   50   60   70   80   90   100%
[----|----|----|----|----|----|----|----|----|----|
**************************************************|
18:31:22 Optimization finished
proc.time()[3] - previous_time
elapsed 
    4.1 
DimPlot(sub_Estrus_new, reduction = "umap") + ggtitle(label = 'Rescale UMAP after removing Immune cells')

FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Crabp2', 'Myh11', 'Wt1', 'S100g', 'Sprr2f',
                  'Fxyd4'))

FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Dcn', 'Pax8', 'Pax2', 'Twist2', 'Igf1', 'Serpina1e', 'Pdgfra', 'Krt19', 'Mki67'))

Marker genes

marker_genes = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4', 'Igfbp6',
                 'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1')

UMAP for marker genes

FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4'))

FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Igfbp6',
                                                             'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1'))

Rename clusters

Cluster.names <- c('1 Secretory_InfAmp',
                   '2 Ciliated_InfAmp',
                   '3 Fibroblast Pdgfra+',
                   '4 Secretory_IsthUTJ',
                   '5 Fibroblast Pdgfra-',
                   '6 Muscle')
names(Cluster.names) <- levels(sub_Estrus_new)
sub_Estrus_new <- RenameIdents(sub_Estrus_new, Cluster.names)
DimPlot(sub_Estrus_new, reduction = "umap", pt.size = 0.5) + scale_color_manual(labels = Cluster.names,values=c('green',                                                                                        'red','blue','orange','grey','yellow','pink'))

DotPlot(sub_Estrus_new, features = marker_genes, group.by = sub_Estrus_new@meta.data$ident) +  
  theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1)) +
  scale_y_discrete(labels = Cluster.names)

Number of cells in each clusters

table(sub_Estrus_new@active.ident, sub_Estrus_new@meta.data$orig.ident)
                      
                       Estrus
  1 Secretory_InfAmp      603
  2 Ciliated_InfAmp       502
  3 Fibroblast Pdgfra+    330
  4 Secretory_IsthUTJ     271
  5 Fibroblast Pdgfra-    129
  6 Muscle                 90

Extract top genes for each cluster

  • Default method of finding highly expressed genes for FindAllMarkers is Wilcoxon Rank Sum test
sub_Estrus_new_markers <- FindAllMarkers(sub_Estrus_new, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25)
Calculating cluster 1 Secretory_InfAmp

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~03s          
  |++                                                | 2 % ~03s          
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  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03s  
Calculating cluster 2 Ciliated_InfAmp

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~10s          
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  |++++++++++++++++++++++++++++                      | 56% ~04s          
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  |+++++++++++++++++++++++++++++                     | 58% ~04s          
  |++++++++++++++++++++++++++++++                    | 59% ~04s          
  |++++++++++++++++++++++++++++++                    | 60% ~04s          
  |+++++++++++++++++++++++++++++++                   | 61% ~04s          
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  |++++++++++++++++++++++++++++++++                  | 63% ~04s          
  |++++++++++++++++++++++++++++++++                  | 64% ~04s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~03s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~03s          
  |++++++++++++++++++++++++++++++++++                | 67% ~03s          
  |++++++++++++++++++++++++++++++++++                | 68% ~03s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~03s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~03s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~03s          
  |++++++++++++++++++++++++++++++++++++              | 72% ~03s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~03s          
  |+++++++++++++++++++++++++++++++++++++             | 74% ~03s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~02s          
  |++++++++++++++++++++++++++++++++++++++            | 76% ~02s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~02s          
  |+++++++++++++++++++++++++++++++++++++++           | 78% ~02s          
  |++++++++++++++++++++++++++++++++++++++++          | 79% ~02s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~02s          
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~02s          
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~02s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 86% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 88% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 90% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 92% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 93% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~01s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=10s  
Calculating cluster 3 Fibroblast Pdgfra+

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~02s          
  |++                                                | 2 % ~02s          
  |++                                                | 3 % ~02s          
  |+++                                               | 4 % ~02s          
  |+++                                               | 5 % ~02s          
  |++++                                              | 6 % ~02s          
  |++++                                              | 8 % ~02s          
  |+++++                                             | 9 % ~02s          
  |+++++                                             | 10% ~02s          
  |++++++                                            | 11% ~02s          
  |++++++                                            | 12% ~02s          
  |+++++++                                           | 13% ~02s          
  |+++++++                                           | 14% ~02s          
  |++++++++                                          | 15% ~02s          
  |+++++++++                                         | 16% ~02s          
  |+++++++++                                         | 17% ~02s          
  |++++++++++                                        | 18% ~02s          
  |++++++++++                                        | 19% ~02s          
  |+++++++++++                                       | 20% ~02s          
  |+++++++++++                                       | 22% ~02s          
  |++++++++++++                                      | 23% ~02s          
  |++++++++++++                                      | 24% ~02s          
  |+++++++++++++                                     | 25% ~02s          
  |+++++++++++++                                     | 26% ~02s          
  |++++++++++++++                                    | 27% ~02s          
  |++++++++++++++                                    | 28% ~02s          
  |+++++++++++++++                                   | 29% ~02s          
  |++++++++++++++++                                  | 30% ~02s          
  |++++++++++++++++                                  | 31% ~02s          
  |+++++++++++++++++                                 | 32% ~02s          
  |+++++++++++++++++                                 | 33% ~02s          
  |++++++++++++++++++                                | 34% ~02s          
  |++++++++++++++++++                                | 35% ~02s          
  |+++++++++++++++++++                               | 37% ~02s          
  |+++++++++++++++++++                               | 38% ~02s          
  |++++++++++++++++++++                              | 39% ~02s          
  |++++++++++++++++++++                              | 40% ~01s          
  |+++++++++++++++++++++                             | 41% ~01s          
  |+++++++++++++++++++++                             | 42% ~01s          
  |++++++++++++++++++++++                            | 43% ~01s          
  |+++++++++++++++++++++++                           | 44% ~01s          
  |+++++++++++++++++++++++                           | 45% ~01s          
  |++++++++++++++++++++++++                          | 46% ~01s          
  |++++++++++++++++++++++++                          | 47% ~01s          
  |+++++++++++++++++++++++++                         | 48% ~01s          
  |+++++++++++++++++++++++++                         | 49% ~01s          
  |++++++++++++++++++++++++++                        | 51% ~01s          
  |++++++++++++++++++++++++++                        | 52% ~01s          
  |+++++++++++++++++++++++++++                       | 53% ~01s          
  |+++++++++++++++++++++++++++                       | 54% ~01s          
  |++++++++++++++++++++++++++++                      | 55% ~01s          
  |++++++++++++++++++++++++++++                      | 56% ~01s          
  |+++++++++++++++++++++++++++++                     | 57% ~01s          
  |++++++++++++++++++++++++++++++                    | 58% ~01s          
  |++++++++++++++++++++++++++++++                    | 59% ~01s          
  |+++++++++++++++++++++++++++++++                   | 60% ~01s          
  |+++++++++++++++++++++++++++++++                   | 61% ~01s          
  |++++++++++++++++++++++++++++++++                  | 62% ~01s          
  |++++++++++++++++++++++++++++++++                  | 63% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 65% ~01s          
  |+++++++++++++++++++++++++++++++++                 | 66% ~01s          
  |++++++++++++++++++++++++++++++++++                | 67% ~01s          
  |++++++++++++++++++++++++++++++++++                | 68% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 69% ~01s          
  |+++++++++++++++++++++++++++++++++++               | 70% ~01s          
  |++++++++++++++++++++++++++++++++++++              | 71% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 72% ~01s          
  |+++++++++++++++++++++++++++++++++++++             | 73% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 74% ~01s          
  |++++++++++++++++++++++++++++++++++++++            | 75% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 76% ~01s          
  |+++++++++++++++++++++++++++++++++++++++           | 77% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 78% ~01s          
  |++++++++++++++++++++++++++++++++++++++++          | 80% ~01s          
  |+++++++++++++++++++++++++++++++++++++++++         | 81% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++         | 82% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++        | 83% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++        | 84% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++       | 85% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 86% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++      | 87% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 88% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++     | 89% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 90% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++    | 91% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 92% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++   | 94% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 95% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++  | 96% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 97% ~00s          
  |+++++++++++++++++++++++++++++++++++++++++++++++++ | 98% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 99% ~00s          
  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=03s  
Calculating cluster 4 Secretory_IsthUTJ

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~04s          
  |++                                                | 2 % ~04s          
  |++                                                | 3 % ~04s          
  |+++                                               | 4 % ~04s          
  |+++                                               | 5 % ~04s          
  |++++                                              | 6 % ~04s          
  |++++                                              | 7 % ~03s          
  |+++++                                             | 8 % ~03s          
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  |++++++                                            | 10% ~03s          
  |++++++                                            | 11% ~03s          
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  |++++++++                                          | 15% ~03s          
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  |++++++++++++++                                    | 27% ~03s          
  |++++++++++++++                                    | 28% ~03s          
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  |+++++++++++++++                                   | 30% ~03s          
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  |++++++++++++++++++                                | 34% ~02s          
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  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=04s  
Calculating cluster 5 Fibroblast Pdgfra-

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~04s          
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  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=04s  
Calculating cluster 6 Muscle

  |                                                  | 0 % ~calculating  
  |+                                                 | 1 % ~02s          
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  |++++++++++++++++++++++++++++++++++++++++++++++++++| 100% elapsed=02s  
sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    slice_max(n = 20, order_by = avg_log2FC)

Heat map for top 10 genes in each cluster

sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    top_n(n = 10, wt = avg_log2FC) -> top10
DoHeatmap(sub_Estrus_new, features = top10$gene) + NoLegend()

---
title: "scRNA-seq analysis using Seurat package"
output: html_notebook
author: Long Nguyen
---
# Description:
* The purpose of this notebook is to analyze scRNA-seq data of mouse Estrus
* The method used is reading directly from fastq files
* The algorithms used come from Seurat package 
* The results will compared to:
  + Same dataset analyzed using Scanpy - read10x
  + Same dataset analyzed using Scanpy - readloom
  
# Workflow begin
This tutorial comes from [Seurat](https://satijalab.org/seurat/articles/pbmc3k_tutorial.html)

## Load required packages
```{r}
library(dplyr)
library(Seurat)
library(leiden)
library(ggplot2)
```


## Load data
Input is a directory that contains fastq files
```{r}
previous_time <- proc.time()[3]
Estrus.data <-Read10X(data.dir ="/home/nguyen/Estrus_cellranger_count/Estrus_Raw/")
proc.time()[3] - previous_time
```
* View(Estrus.data) - run this command to investigate the output
* Current dataset:
  + 31253 genes
  + 6599 cells

## Initialize the Seurat object
* Initialize the Seurat object with the raw (non-normalized data).
* Filter out genes that are detected in less than 3 cells 
* Filter out cells that have less than 200 genes expressed
```{r}
previous_time <- proc.time()[3]
Estrus <- CreateSeuratObject(counts = Estrus.data, project = "Estrus", min.cells = 3, min.features = 200)
proc.time()[3] - previous_time
Estrus
```
*Output:
  + 19420 genes 
  + 6481 cells

## Calculate the percentage of mitochondrial genes
* Add "percent.mt" column to object metadata
* Percentage of mitochondrial genes is stored in "percent.mt"
* "^mt-" means strings start with mt (lowercase mt for mice, uppercase MT for human)
```{r}
Estrus[["percent.mt"]] <- PercentageFeatureSet(Estrus, pattern = "^mt-")
```

```{r}
# This is the list of genes that start with mt
mito_genes <- grep("^mt-", rownames(Estrus) , ignore.case=T, value=T)
print(mito_genes)
```

```{r}
# This is the data in "percent.mt" column
percent_mito <- FetchData(object = Estrus, vars = "percent.mt") #how to fetch data
head(percent_mito)
```

```{r, fig.width=10}
# Visualize QC metrics as a violin plot
VlnPlot(Estrus, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3)
```
```{r}
# FeatureScatter is typically used to visualize feature-feature relationships, but can be used
# for anything calculated by the object, i.e. columns in object metadata, PC scores etc.
plot1 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "percent.mt")
plot2 <- FeatureScatter(Estrus, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
plot1 + plot2
```

## Filter out low-quality cells
* We filter out cells that have unique feature counts over 6000
* Because any cell that has more than 6000 genes are likely doublets
* Note: Seurat tutorial used 2500 instead of 6000
* We also filter out cells that have more than 5% mitochondrial genes
* Because those are likely to be nonviable cells
```{r}
previous_time <- proc.time()[3]
Estrus <- subset(Estrus, subset = nFeature_RNA < 6000 & percent.mt < 5)
proc.time()[3] - previous_time
```
```{r}
Estrus
```
* After filtering step, the dataset is left with
 + 19420 genes
 + 2174 cells

## Normalizing the data
* Perform log-normalization method
* Feature counts for each cell are divided by the total counts for that cell and multiplied by the scale.factor
* scale.factor default value = 10,000
* This is then natural-log transformed using log1p
```{r}
previous_time <- proc.time()[3]
Estrus <- NormalizeData(Estrus, normalization.method = "LogNormalize", scale.factor = 10000)
proc.time()[3] - previous_time
```
## Identification of highly variable features (feature selection)
```{r}
previous_time <- proc.time()[3]
Estrus <- FindVariableFeatures(Estrus, selection.method = "disp", nfeatures = 5000)
proc.time()[3] - previous_time
```
## Plotting highly variable features
```{r}
# Identify the 10 most highly variable genes
top10 <- head(VariableFeatures(Estrus), 10)
top10
```

```{r, fig.width=10,fig.height=4.5}
# Plot variable features with and without labels
plot1 <- VariableFeaturePlot(Estrus)
plot2 <- LabelPoints(plot = plot1, points = top10, repel = TRUE)
plot1 + plot2 
```
## Regress out data and perform scaling
* Regressing out total counts per cell and percent.mt before scaling. 
* Take out mitochondrial genes before scaling (analyzing) the data 
* Because mito_genes shouldn't be accounted for cell population analysis.
```{r}
previous_time <- proc.time()[3]
all.genes <- rownames(Estrus)
Estrus <- ScaleData(Estrus, features = all.genes, vars.to.regress = c("nCount_RNA","percent.mt"), scale.max = 10)
# Explanations below are from [Seurat](https://satijalab.org/seurat/archive/v3.0/cell_cycle_vignette.html)
# vars.to.regress = RegressOut in Seurat version 1.4 or lower
# However, as the results of this procedure are stored in the scaled data slot (therefore overwriting the output of ScaleData) 
# This functionality is merged into the ScaleData function itself.
proc.time()[3] - previous_time
```
## Perform linear dimensional reduction (PCA)
```{r}
previous_time <- proc.time()[3]
Estrus <- RunPCA(Estrus, features = VariableFeatures(object = Estrus))
proc.time()[3] - previous_time
```

## Examine and visualize PCA results a few different ways
```{r}
print(Estrus[["pca"]], dims = 1:5, nfeatures = 5)
```

```{r}
VizDimLoadings(Estrus, dims = 1:2, reduction = "pca")
```

```{r}
DimPlot(Estrus, reduction = "pca") + ggtitle(label = 'Including immune cells')
```

```{r, fig.width=12}
FeaturePlot(Estrus, reduction = "pca", features = c('Ovgp1', 
                                                    'Foxj1', 'Igf1', 'Pdxk', 
                                                    'Fxyd4', 'Bsg', 
                                                    'Serpina1e', 'Krt19'))
```
```{r, fig.width=12}
FeaturePlot(Estrus, reduction = "pca", features = c('Dcn', 'Pax8',
                                                    'Ephx2', 
                                                    'Pecam1', 'Pdgfra', 'Myh11', 'Atp2b4', 
                                                    'Cd52'))
```

## Determine the ‘dimensionality’ of the data
* Seurat tutorial uses JackStraw() and and ScoreJackStraw to determine dimensionality
* the syntax is: 
  + Estrus <- JackStraw(Estrus, num.replicate = 100)
  + Estrus <- ScoreJackStraw(Estrus, dims = 1:20)
* However, it takes very long. 
* An elbow plot often corresponds well enough with the significant dims and is much faster to run than Jackstraw
* According to the graph below, choose the first 37 principle components to use
```{r}
ElbowPlot(Estrus, ndims = 40)
```
## Find neighbors
* Dims = dimensions = determined as 37 from the above step
```{r}
previous_time <- proc.time()[3]
Estrus_new <- FindNeighbors(Estrus, dims = 1:37, k.param = 10)
proc.time()[3] - previous_time
```
## Find cell clusters using Leiden's method
```{r}
# Algorithm = 4 specifies Leiden's method
previous_time <- proc.time()[3]
Estrus_new <- FindClusters(Estrus_new, resolution = 0.12, algorithm = 4)
proc.time()[3] - previous_time
```
## Constructing UMAP plot
```{r}
previous_time <- proc.time()[3]
Estrus_new <- RunUMAP(Estrus_new, dim = 1:37) 
proc.time()[3] - previous_time
```
## Visualization
```{r}
DimPlot(Estrus_new, reduction = "umap") + ggtitle(label = 'Including immune cells')
```
```{r, fig.width=12}
FeaturePlot(Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Dcn', 'Serpina1e', 'Sprr2f',
                                                         'Fxyd4', 'Ly6a', 'Epcam', 'Krt19', 'Vim', 'Pdgfra'))
```

```{r, fig.width=12}
FeaturePlot(Estrus_new, reduction = "umap", features = c('Myh11', 
                                        'Dkk2', 'Slitrk6', 'Dio2', 'Smoc2', 'Tes', 'Dab1', 'Camk1d', 'Pecam1', 'Cd52','Mki67'))

```
## Removing immune cells
```{r, fig.width=12}
sub_Estrus = subset(x = Estrus_new, ident = c(1,2,3,4,5,7,8,9,10,12))
DimPlot(sub_Estrus, reduction = "umap") + ggtitle(label = 'Immune cells removed')
```
```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Fxyd4', 'Cxcl17', 'Myh11', 'Cd52', 'Twist2', 'Pecam1'))
```
```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "umap", features = c('Pdgfra', 'Ephx2', 'Dcn', 'Krt19', 'Epcam', 'Vim','Mki67'))
```
```{r}
sub_Estrus
```
* After removing immune cells, the dataset is left with:
  + 19420 genes
  + 1925 cells

## Re-run analysis after excluding immune cells
```{r}
previous_time <- proc.time()[3]
sub_Estrus <- RunPCA(sub_Estrus, features = VariableFeatures(object = sub_Estrus))
proc.time()[3] - previous_time
```

```{r, fig.width=12}
DimPlot(sub_Estrus, reduction = "pca") + ggtitle(label = 'Immune cells removed')
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "pca", features = c('Esr1', 'Ovgp1', 'Foxj1', 'Fxyd4', 'Ephx2', 'Dcn'))
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus, reduction = "pca", features = c('Fos', 'Kcne3', 'Serpina1e', 'Crabp2', 'Pgr', 'Vim'))
```
```{r}
previous_time <- proc.time()[3]
sub_Estrus_new <- FindNeighbors(sub_Estrus, dims = 1:37)
proc.time()[3] - previous_time
```

```{r}
previous_time <- proc.time()[3]
sub_Estrus_new <- FindClusters(sub_Estrus_new, resolution = 0.045, algorithm = 4)
proc.time()[3] - previous_time
```

```{r}
previous_time <- proc.time()[3]
sub_Estrus_new <- RunUMAP(sub_Estrus_new, dim = 1:37)
proc.time()[3] - previous_time
```

```{r, fig.width=10}
DimPlot(sub_Estrus_new, reduction = "umap") + ggtitle(label = 'Rescale UMAP after removing Immune cells')
```
```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Ovgp1', 'Foxj1', 'Crabp2', 'Myh11', 'Wt1', 'S100g', 'Sprr2f',
                  'Fxyd4'))
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Dcn', 'Pax8', 'Pax2', 'Twist2', 'Igf1', 'Serpina1e', 'Pdgfra', 'Krt19', 'Mki67'))
```
## Marker genes
```{r}
marker_genes = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4', 'Igfbp6',
                 'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1')
```

## UMAP for marker genes
```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Epcam', 'Krt15','Krt8','Krt19','Vim','Pdgfra','Twist2', 'Sprr2f', 'Fxyd4'))
```

```{r, fig.width=12}
FeaturePlot(sub_Estrus_new, reduction = "umap", features = c('Igfbp6',
                                                             'Acta2', 'Myh11','Foxj1','Ovgp1','Wt1','Serpina1e','S100g','Pecam1'))
```

## Rename clusters
```{r}
Cluster.names <- c('1 Secretory_InfAmp',
                   '2 Ciliated_InfAmp',
                   '3 Fibroblast Pdgfra+',
                   '4 Secretory_IsthUTJ',
                   '5 Fibroblast Pdgfra-',
                   '6 Muscle')
names(Cluster.names) <- levels(sub_Estrus_new)
sub_Estrus_new <- RenameIdents(sub_Estrus_new, Cluster.names)
DimPlot(sub_Estrus_new, reduction = "umap", pt.size = 0.5) + scale_color_manual(labels = Cluster.names,values=c('green',                                                                                        'red','blue','orange','grey','yellow','pink'))
```
```{r, fig.width=12}
DotPlot(sub_Estrus_new, features = marker_genes, group.by = sub_Estrus_new@meta.data$ident) +  
  theme(axis.text.x = element_text(angle = 90, vjust = 0.5, hjust=1)) +
  scale_y_discrete(labels = Cluster.names)
```
## Number of cells in each clusters
```{r}
table(sub_Estrus_new@active.ident, sub_Estrus_new@meta.data$orig.ident)
```

## Extract top genes for each cluster
* Default method of finding highly expressed genes for FindAllMarkers is Wilcoxon Rank Sum test
```{r}
sub_Estrus_new_markers <- FindAllMarkers(sub_Estrus_new, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25)
sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    slice_max(n = 20, order_by = avg_log2FC)
```
## Heat map for top 10 genes in each cluster
```{r, fig.width=10}
sub_Estrus_new_markers %>%
    group_by(cluster) %>%
    top_n(n = 10, wt = avg_log2FC) -> top10
DoHeatmap(sub_Estrus_new, features = top10$gene) + NoLegend()
```

